Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

Depolarization induced by injection of cyclic nucleotides into frog taste cell

In order to identify the intracellular transmitter involved in the taste transduction process, cyclic nucleotides were iontophoretically injected into the frog taste cells while membrane potentials were recorded intracellularly. Injection of either cyclic GMP or cyclic AMP induced a depolarization response of about 5 mV in the taste cells, but injection of Cl- had no effect. The rate of a repolarization after the depolarization elicited by cyclic GMP was larger than that after cyclic AMP. The possible role of cyclic nucleotide in the taste transduction was discussed.     

Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or Rous sarcoma virus-induced transformed cells

With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein.     

Transforming c-Ki-ras mutation is a preneoplastic event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea.

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35----A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.     

Dependence on exercise intensity of changes in electrolyte secretion from the skin sampled by a simple method

Secreta from the palm and forearm was sampled for 1-min periods by a new technique, using a glass cylinder. Subjects exercised for 10-min periods at successive intensities of 40%, 50% and 65% VO2max with a leg ergometer operated in the supine position. Changes in the concentrations (values) of Na+, K+ and Cl- in their secreta during exercise were investigated. Significant positive correlations were found between the values of any two electrolytes in samples from the palm or the forearm, but the correlations between values for any one of the three electrolytes from the two sites were not significant. Values for concentrations of the electrolytes were significantly higher in samples from the palm than in those from the forearm at rest, 10 min after the beginning of exercise and at the end of exercise. No significant correlation was found between values for electrolytes in samples from the palm and the exercise intensity, but values for Na+ in samples from the forearm increased stepwise with increase in exercise intensity, and similar tendencies were observed for values of K+ and Cl-. The values for the three electrolytes in samples from the forearm, but not the palm, were significantly correlated with values for blood lactate, the percentage of VO2max and the heart rate. These results suggest that the present technique is suitable for successive samplings of secreta from the forearm, and that values for the electrolytes in samples are useful indices of exercise intensity.     

Nucleotide sequence of the LuxC gene and the upstream DNA from the bioluminescent system of Vibrio harveyi.

The nucleotide sequence of the luxC gene (1431 bp) and the upstream DNA (1049 bp) of the luminescent bacterium Vibrio harveyi has been determined. The luxC gene can be translated into a polypeptide of 55 kDa in excellent agreement with the molecular mass of the reductase polypeptide required for synthesis of the aldehyde substrate for the bioluminescent reaction. Analyses of codon usage showed a high frequency (1.9%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA, B and D genes. The low G/C content of the luxC gene and upstream DNA (38-39%) compared to that found in the other lux genes of V. harveyi (45%) was primarily due to a stretch of 500 nucleotides with only a 24% G/C content, extending from 200 bp inside lux C to 300 bp upstream. Moreover, an open reading frame did not extend for more than 48 codons between the luxC gene and 600 bp upstream at which point a gene transcribed in the opposite direction started. As the lux system in the luminescent bacterium, V. fischeri, contains a regulatory gene immediately upstream of luxC transcribed in the same direction, these results show that the organization and regulation of the lux genes have diverged in different luminescent bacteria.     

Two-dimensional flow cytometric analysis of T cell subsets maintained in IL-2 medium after autologous mixed leukocyte reaction activation

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin-2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two-dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin-2, there was a progressive increase in the percentages of CD8+ Leu 15- cytotoxic T, CD4+ Leu 8- helper T, and CD3+ HLA-DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)-rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock-T cells and ammonium chloride-T cells in the phenotypic changes of T cell subsets after long-term culture in an interleukin-2 medium may suggest a regulatory role of the ammonium chloride-sensitive T cells in the AMLR.